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Scientists have demonstrated the feasibility of correcting a disease-causing genetic mutation by generating iPSCs from patient fibroblasts, precisely cutting out the mutant transcript and pasting in the correct version, and then differentiating the genetically corrected iPSCs into the cell type required for autologous therapy.

An international team led by researchers at the Wellcome Trust Sanger Institute and University of Cambridge in the U.K. used a combination of zing finger nucleases and piggyBac transposon technology to carry out biallelic correction of the point mutation responsible for α1-antitrypsin deficiency (A1ATD) in iPSCs derived from A1ATD patients. Importantly, using this technique leaves no residual nonhuman sequences in the corrected cells. When the genetically corrected iPSCs were differentiated into hepatocyte-like cells and transplanted into experimental mice, they integrated into the liver and led to the production of serum albumin.

The researchers claim that, as far as they are aware, the results provide the first proof of principal for the potential of combining human iPSCs with genetic correction to generate clinically relevant cells for autologous cell-based therapies. Their work is reported in Nature in a paper titled “targeted gene correction of α1-antitrypsin deficiency in induced pluripotent stem cells.” The studies were led by Kosuke Yusa, Ph.D., and Allan Bradley, Ph.D., at the Wellcome Trust Sanger Institute, together with David A. Lomas, Ph.D., at the Cambridge Institute for Medical Research (University of Cambridge), and S. Tamir Rashid, Ph.D., and Ludovic Vallier, Ph.D., at the University of Cambridge Anne McLaren Laboratory for Regenerative Medicine.

Current approaches to gene targeting are based on positive selection to isolate clones that have undergone homologous recombination. Removing the unwanted selection cassettes then involves the use of Cre/loxP or Flp/FRT recombination systems, the authors explain. However, this leaves behind single loxP or FRT sites, and retention of the small ectopic sequences could potentially interfere with transcriptional regulatory elements of surrounding genes.

An alternative method for removing selection cassettes is to convert them into transposons, and the moth-derived piggyBac DNA transposon is ideally suited to this task, as it can transpose efficiently in mammalian cells including human embryonic stem (ES) cells, they continue. One of the major benefits of piggyback mobile elements is that the technology enables the removal of transgenes flanked by piggyBac inverted repeats without leaving any residual sequences.

To see how well piggyBac worked with iPSCs, the team initially used the system to correct the point mutation responsible for albinism (G290T substitution in the Tyr) in iPSCs generated from fibroblasts taken from C57Bl6-Tyrc-Brd animals. The targeting vector was constructed to carry a wild-type 290G sequence and a PGK-puroDtk cassette, flanked by piggyBac repeats into the TTAA site. After isolating targeted clones, the selection cassette was excised from the iPSC genome by transient expression of piggyBac transposase, and subsequent 1-(2-deoxy-2-fluoro-b-D-arabinofuranosyl)-5-iodouracil (FIAU) selection. Correction of the G290T mutation and seamless piggyBac excision were confirmed by sequence analyses. When iPSCs with the reverted allele were injected into albino mouse blastocysts, the resulting chimeric mice had a black coat color, confirming phenotypic correction of the albino mutation.

The researchers then moved on to see whether the same technology could be used to correct a mutation in human iPSCs derived from individuals with A1ATD, the most common inherited metabolic disease of the liver. A1ATD is caused by a single point mutation in the α1-antitrypsin (A1AT) gene, and causes the protein to form polymers within hepatocyte endoplasmic reticulum, which leads to cirrhosis.

Given that homologous recombination is relatively inefficient in human ES cells, the researchers used zinc finger nuclease (ZFN) technology to prompt gene targeting. The ZFN pairs were designed to cleave the site of the A1AT Z mutation in affected cells, and a targeting vector was constructed from isogenic DNA with piggyBac repeats flanking the PGK-puroDtk cassette. A1ATD-iPSC lines derived from three different patients generated targeted clones. Fifty-four percent of these were targeted on just one allele, whereas simultaneous targeting of both alleles occurred in 4%. Removal of the piggyBac-flanked selection cassette from two of these homozygously targeted clones was then carried out by tranfection with a hyperactive form of the piggyBac transposase.

Genetic analysis indicated that biallelic excision had successfully been effected in 11% of the resulting colonies, and confirmed that the Z mutation had been corrected on both alleles. The corrected iPSC lines were capable of maintaining pluripotency markers for over 20 passages, and were able to differentiate into cells expressing markers of all three germ layers, which suggested that correction of the A1AT mutation hadn’t affected pluripotency.

The investigators in addition carried out comparative genome hybridization (CGH) to check whether either the initial iPSC cell lines generated from the A1ATD patients, and/or the genetically corrected iPSCs, had developed genomic instability. They found that two of three primary A1ATD-iPSC lines did, indeed, display DNA amplifications or deletions that weren’t present in the parental fibroblast cell lines. However, they also found that after ZFN-stimulated targeting, four of the six homozygous clones showed no evidence of genome alteration in comparison with their parental iPSC lines. Moreover, 12 of 16 cell lines with biallelic piggyBac excision had unaltered genomes when compared with their corresponding primary iPSCs.

SNP array analyses similarly found no evidence of loss of heterozygosity. “This observation demonstrates that biallelic gene correction was the result of simultaneous homologous recombination followed by simultaneous excision at both alleles, and that mitotic recombination was not involved in this process,” the authors stress.

The next stage was to evaluate the effects of genetically corrected iPSCs in vivo. The cells were differentiated in vitro into hepatocyte-like cells, which the team assessed using CGH to confirm that no genetic abnormalities had crept in during the differentiation process. As well as being genetically normal, hepatocyte-like cells differentiated from the corrected iPSCs demonstrated the same functional features as native hepatocytes, including glycogen storage, low density lipoprotein (LDL)-cholesterol uptake, albumin secretion, and cytochrome P450 activity.

Significantly, the researchers point out, ELISA analysis confirmed that there was no mutant polymeric A1AT in the iPSC-derived hepatocyte-like cells, but rather the cells efficiently secreted normal endoglycosidase-H-insensitive monomeric A1AT, which showed enzymatic inhibitor activity comparable to that produced by normal native hepatocytes.

The corrected iPSC-derived hepatocyte-like cells were then transplanted into livers of Alb-uPA+/+;Rag2-/-;Il2rg-/- mice. Evaluation of the livers from treated mice 14 days later confirmed that the human hepatocyte-like cells were distributed throughout the liver lobes and were also integrated into the existing mouse parenchyma. Human albumin was found in the serum of transplanted animals for at least five weeks, and none of the treated animals developed tumors.

“Therefore, corrected iPSC-derived hepatocyte-like cells were able to colonize the liver in vivo and show functional activities characteristic of their human ES-cell-derived counterparts,” the investigators state.

They finally replicated their work using cells that represented a clinically more relevant evaluation of the technique. To this end, they generated A1ATD iPSCs by reprogramming patients’ own fibroblasts with Sendai virus vectors. Genomically intact primary iPSCs that resulted from the reprogramming stage were corrected for A1AT deficiency using the same ZFN/piggyBac technique employed to correct the gene in the test cell lines. The final product was, as hoped, a cell line that carried the corrected A1AT, demonstrated an intact genome compared with its parental fibroblast, and expressed normal A1AT protein when differentiated into hepatocyte-like cells.

“This is the first demonstration, to our knowledge, of the generation of mutation-corrected patient-specific iPSCs, which could realize the therapeutic promise of human iPSCs,” the investigators conclude. “iPSCs derived from different patients were effectively corrected, demonstrating that this method could be applied to a large number of A1ATD-iPSC lines. Because the biallelic correction could be carried out in less than four months, our approach may be compatible with large-scale production of corrected patient-specific iPSCs not only for A1ATD but also for other monogenic disorders.”

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